PROJECT SUMMARY Red blood cell (RBC) transfusion is costly, and is among the procedures most frequently performed in VA and other health care settings. Growing evidence, however, suggests that many anemic patients may not benefit from RBC transfusion, and that blood function declines even during short storage periods. We have identified a novel mechanism whereby RBCs normally export nitric oxide (NO)-derived signals to the vasculature, facilitating the flow of blood itself. S-nitrosothiols (SNOs) are thiol adducts formed in RBCs from precursor NO in concert with the oxygenation-linked allosteric transition in hemoglobin. RBCs export these vasoregulatory SNOs ?on demand? in order to fine-tune regional blood flow and prevent RBC adhesion to the endothelium (ECs). Preliminary Studies show for the first time that intercellular transport of small carrier SNOs is critical in the prevention of RBC adhesion to ECs. These novel findings demonstrate the role of blood flow-regulating, intercellular SNO signaling by the RBC, and impairment in this function after storage. We will test the hypothesis that SNO transport is critical to the vasoregulatory function of RBCs, and its loss a remediable ?storage lesion? promoting post-transfusion lung morbidity, by accomplishing these Specific Aims: Aim 1: Determine the role of RBC LAT1 in mediating SNO export by human RBCs. The specific conduit by which RBCs export SNO groups remains undetermined, but our Preliminary Studies indicate a significant role of RBC LAT1 in SNO export. We will use new tools and pharmacological approaches to determine the role of LAT1 in RBC SNO export, and measures to augment this activity. We will also identify intracellular RBC molecular targets of beneficial SNO restoration. Aim 2: Determine the role of SNO import via LAT1 in endothelial cells in limiting the adhesivity of healthy human RBCs. We showed that SNO can be restored in banked RBCs after storage-induced loss, and that SNO restoration limits RBC-EC adhesion. We will determine whether LAT1-mediated SNO import by ECs is necessary for the RBC SNO-induced antiadhesive effect by using genetic and pharmacological approaches, and test the role of alternative mechanisms. Aim 3: Determine the role of LAT1 in the storage-sensitive effects of RBC SNOs in mouse models of human RBC transfusion. We developed an innovative mouse model of human-RBC transfusion, recapitulating key clinical phenotypes such as lung dysfunction. We will use intravital microscopy, a novel mouse conditionally deficient in EC LAT1, and transfusate labeling to determine the role of RBC SNO in limiting post-transfusion morbidity, specifically RBC adhesion and depressed blood oxygenation and tissue O2 delivery. Both nave and two-hit (transfusion after lipopolysaccharide), acute and chronic models will be used. Determining the role of SNO transport in signaling from RBCs to ECs in health and disease is the next logical step toward our long- term scientific objective: to understand the mechanisms whereby RBCs export mediators to regulate their own flow in response to tissue needs. These studies are expected to inform changes in transfusion practice to improve the risk-benefit balance for Veteran patients.